Gram-positive bacteria are surrounded by a cell wall containing polypeptides and polysaccharide. The gram-positive cell wall appears as a broad, dense wall that is 20-80 nm thick and consists of numerous interconnecting layers of peptidoglycan. Between 60% and 90% of the gram-positive cell wall is peptidoglycan, providing cell shape, a rigid structure, and resistance to osmotic shock. The cell wall does not exclude the Gram stain crystal violet, allowing cells to be stained purple, and therefore “Gram-positive.” The peptidoglycan molecule's backbone is comprised of glucose derivatives N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), interconnected by peptides. Interwoven in the peptidoglycan cell wall are teichoic acids and lipoteichoic acids. The gram-positive peptidoglycan is studded with surface proteins, including enzymes, invasins, adhesins and other binding proteins.
Gram-positive bacteria include but are not limited to the genera Actinomyces, Bacillus, Listeria, Lactococcus, Staphylococcus, Streptococcus, Enterococcus, Mycobacterium, Corynebacterium, and Clostridium. Medically relevant species include Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, and Enterococcus faecalis. Bacillus species, which are spore-forming, cause anthrax and gastroenteritis. Spore-forming Clostridium species are responsible for botulism, tetanus, gas gangrene and pseudomembranous colitis. Corynebacterium species cause diphtheria, and Listeria species cause meningitis.
The cell walls of gram-negative bacteria are more chemically complex, thinner and less compact. In gram-negative bacteria, peptidoglycan makes up 5-20% of the cell wall and is not the outermost layer, lying between the plasma membrane and an outer membrane. The outer membrane is composed of lipopolysaccharide (LPS) which is an endotoxin. The LPS prevents penetration of gram stain, rendering these bacteria “gram negative.” Gram-negative and Gram-positive bacteria can be susceptible to distinct antibacterial agents and therapeutic molecules.
Antibacterials that inhibit cell wall synthesis, such as penicillins and cephalosporins, interfere with the linking of the interpeptides of peptidoglycan and weaken the cell wall of both gram positive and gram negative bacteria. Because the peptidoglycans of gram-positive bacteria are exposed, gram-positive bacteria are more susceptible to these antibiotics. Advantageously, eukaryotic cells lack cell walls and are not susceptible to these drugs or other cell wall agents.
Thus, in gram-positive bacteria, the cell membrane is surrounded by a cell wall containing polypeptides and polysaccharide that is 20-80 nm thick and consists of numerous interconnecting layers of peptidoglycan. The cell membrane carries out multiple functions and contains enzymes of biosynthetic pathways for synthesis of cell wall phospholipids, peptidoglycans, etc. The cell membrane also contains carrier proteins, transport proteins, and permeases for transport of organics and inorganics across the cell membrane. Components for control of chemotaxis are located in the cell membrane. Gram positive cell membrane protein families include penicillin binding proteins, ABC transporters, and potassium channels.
Gram-positive surface proteins are attached to the cell wall and displayed on the surface via a mechanism involving the enzyme(s) sortase. The genomes of most gram-positive bacteria encode two or more sortase enzymes, which have different sorting motif target sequences. The most common sorting target sequence is an LPXTG (SEQ ID NO: 4) motif. The sorting mechanism was first characterized in S. aureus, where the srtA (surface protein sorting A) gene was identified as restoring the defect in cell wall anchoring of Protein A (Mazmanian, S. K. et al (1999) Science 285:760-763; Ton-That, H. et al (1999) PNAS 96(22):12424-12429). Protein A is an S. aureus surface protein and is synthesized as a precursor with an N-terminal signal peptide and a C-terminal sorting signal, an LPXTG (SEQ ID NO: 4) motif (Schneewind, O. et al (1992) Cell 70:267-281). The Protein A sorting signal directs the peptide to the cell wall envelope and it is then cleaved between the threonine and the glycine of the LPXTG (SEQ ID NO: 4) sequence. The S. aureus sortase B anchors iron-regulated surface determinant C (IsdC), which has an NPQTN motif sorting signal (Marraffini, L. A. et al (2004) J Biol Chem 279:37763-37770). Streptococcal SrtC2 recognizes surface proteins with QVPTGV (SEQ ID NO: 5) motif signals (Barnett, T. C. et al (2004) J Bact 186:5865-5875).
The S. aureus sortase SrtA is a cell membrane-anchored enzyme and has been demonstrated to be absolutely required for the anchoring of S. aureus surface proteins to the cell wall envelope and essential for pathogenesis of animal infections (Mazmanian, S. K. et al (2000) PNAS 97(10):5510-5515; Cossart, P and Jonquieres, R. (2000) PNAS 97(10):5013-5015). In these studies, the functional assembly of all staphylococcal adhesins, protein A, fibronectin-binding proteins (FnbA and FnbB) and clumping factors (ClfA and ClfB) was abolished in sortase SrtA mutants. Sortase SrtA cleaves surface protein precursors between the threonine and the glycine of the LPXTG (SEQ ID NO: 4) motif and then captures the C-terminal carboxyl by formation of a thioester bind with its active sulfhydryl. The sortase then completes the transpeptidation reaction via nucleophilic attack of the amino group of the lipid II peptidoglycan precursor, forming an amide bond between the surface peptide and cell wall cross bridge and regenerating its active site sulfhydryl (Ton-That, H and Schneewind, O. (1999) J. Biol Chem 274:24316-24320)
Scientific studies point to sortase and sortase family members as playing a universal role in gram-positive bacteria. Surface proteins with C-terminal LPXTG (SEQ ID NO: 4) motifs have been found in all pathogenic gram-positive bacteria (Navarre, W. W. et al (1999) Microbiol Mol Biol Rev 63:174-229). Sortase homologs have been identified in each of and various Bacillus, Enterococcus, Actinomyces, Lactococcus, Listeria, Clostridium and Corynebacterium (Mazmanian, S. K. et al (1999) Science 285:760-763; Navarre, W. W. and Schneewind, O. (1999) Microbiol Mol Biol Rev 63:174-229). In murine models of organ abscesses, infectious arthritis, and endocarditis, Staphylococcal sortase srtA mutants display significant defects in pathogenesis (Jonsson, I. M. et al (2002) J Infect Dis 185:1417-1424; Jonsson, I. M. et al (2003) Microb Infect 5:775-780).
Fractionation and Western blotting experiments using recombinant sortase antibodies have shown that sortase is a bacterial membrane associated protein (Mazmanian, S. K. et al (2000) RNAS 97(10):5510-5515). For these experiments, S. aureus cultures were fractionated into extra cellular medium, cell wall digest, cytosol, and membrane compartments, and sortase was found only in the membrane. Immunoblotting with sortase-specific antibodies shows that the sortase is not removed by treatment of Staphylococci with trypsin (De Dent, A. C. et al (2007) J Bact 189(12):4473-4484). While the particular distribution of many surface proteins, including proteins in the bacterial membrane, has been determined, the actual or precise distribution of sortase A in the membrane is not readily determinable, including in S. aureus for instance. Under ordinary growth and standard culture conditions, proteases and antibodies have limited accessibility to sortase on the bacterial surface (De Dent A. C. et al (2007) J Bact 189(12):4473-4484). Recent studies have confirmed its location at the cell membrane using fixed cells (Raz A and Fischetti V A (2008) PNAS 105(47):18549-18554).
It is apparent that antibody(ies) or other large molecules and macromolecules (for example protease) cannot readily access their cell membrane targets without crossing the cell wall and peptidoglycan layer. Thus, for instance, the cell membrane protein sortase is not ideally susceptible as a target for directed therapy, including antibody therapy. Thus, cell membrane embedded and/or associated proteins, or other proteins below or within the gram positive bacterial cell wall, are not readily accessible and ideally susceptible to certain therapeutic intervention due to the cell wall and the thick peptidoglycan. Therefore, in view of the limited accessibility of gram-positive cell membrane proteins, such as sortase, it should be apparent that there still exists a need in the art for methods, approaches and therapeutic compositions to permit effective targeting and modulation of bacterial cell membrane proteins, such as sortase, including by antibodies or other enzymes or macromolecules.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.